Centrosomes: The good and the bad for brain development.

Centrosomes nucleate and organise the microtubule cytoskeleton in animal cells. These membraneless organelles are key structures for tissue organisation, polarity and growth. Centrosome dysfunction, defined as deviation in centrosome numbers and/or structural integrity, has major impact on brain size and functionality, as compared with other tissues of the organism. In this review, we discuss the contribution of centrosomes to brain growth during development. We discuss in particular the impact of centrosome dysfunction in Drosophila and mammalian neural stem cell division and fitness, which ultimately underlie brain growth defects.

Cell-Cycle Asynchrony Generates DNA Damage at Mitotic Entry in Polyploid Cells.

Polyploidy arises from the gain of complete chromosome sets [1], and it is known to promote cancer genome evolution. Recent evidence suggests that a large proportion of human tumors experience whole-genome duplications (WGDs), which might favor the generation of highly abnormal karyotypes within a short time frame, rather than in a stepwise manner [2-6]. However, the molecular mechanisms linking whole-genome duplication to genetic instability remain poorly understood. Using repeated cytokinesis failure to induce polyploidization of Drosophila neural stem cells (NSCs) (also called neuroblasts [NBs]), we investigated the consequences of polyploidy in vivo. Surprisingly, we found that DNA damage is generated in a subset of nuclei of polyploid NBs during mitosis. Importantly, our observations in flies were confirmed in mouse NSCs (mNSCs) and human cancer cells after acute cytokinesis inhibition. Interestingly, DNA damage occurs in nuclei that were not ready to enter mitosis but were forced to do so when exposed to the mitotic environment of neighboring nuclei within the same cell. Additionally, we found that polyploid cells are cell-cycle asynchronous and forcing cell-cycle synchronization was sufficient to lower the levels of DNA damage generated during mitosis. Overall, this work supports a model in which DNA damage at mitotic entry can generate DNA structural abnormalities that might contribute to the onset of genetic instability.

Differences in Mitotic Spindle Architecture in Mammalian Neural Stem Cells Influence Mitotic Accuracy during Brain Development.

A functional bipolar spindle is essential to segregate chromosomes correctly during mitosis. Across organisms and cell types, spindle architecture should be optimized to promote error-free divisions. However, it remains to be investigated whether mitotic spindle morphology adapts to changes in tissue properties, typical of embryonic development, in order to ensure different tasks, such as spindle positioning and chromosome segregation. We have characterized mitotic spindles in neural stem cells (NSCs) of the embryonic developing mouse neocortex. Surprisingly, we found a switch in spindle morphology from early to late neurogenic stages, which relies on an increase in inner spindle microtubule density and stability. Mechanistically, we identified the microtubule-associated protein TPX2 as one determinant of spindle shape, contributing not only to its robustness but also to correct chromosome segregation upon mitotic challenge. Our findings highlight a possible causal relationship between spindle architecture and mitotic accuracy with likely implications in brain size regulation.

Chromosome structural anomalies due to aberrant spindle forces exerted at gene editing sites in meiosis.

Mouse female meiotic spindles assemble from acentriolar microtubule-organizing centers (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOC biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master microtubule-organizing center regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules significantly diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.

Centrosome amplification disrupts renal development and causes cystogenesis.

Centrosome number is tightly controlled to ensure proper ciliogenesis, mitotic spindle assembly, and cellular homeostasis. Centrosome amplification (the formation of excess centrosomes) has been noted in renal cells of patients and animal models of various types of cystic kidney disease. Whether this defect plays a causal role in cystogenesis remains unknown. Here, we investigate the consequences of centrosome amplification during kidney development, homeostasis, and after injury. Increasing centrosome number in vivo perturbed proliferation and differentiation of renal progenitors, resulting in defective branching morphogenesis and renal hypoplasia. Centrosome amplification disrupted mitotic spindle morphology, ciliary assembly, and signaling pathways essential for the function of renal progenitors, highlighting the mechanisms underlying the developmental defects. Importantly, centrosome amplification was sufficient to induce rapid cystogenesis shortly after birth. Finally, we discovered that centrosome amplification sensitized kidneys in adult mice, causing cystogenesis after ischemic renal injury. Our study defines a new mechanism underlying the pathogenesis of renal cystogenesis, and identifies a potentially new cellular target for therapy.

Transient PLK4 overexpression accelerates tumorigenesis in p53-deficient epidermis.

Aneuploidy is found in most solid tumours, but it remains unclear whether it is the cause or the consequence of tumorigenesis. Using Plk4 overexpression (PLK4OE) during epidermal development, we assess the impact of centrosome amplification and aneuploidy on skin development and tumorigenesis. PLK4OE in the developing epidermis induced centrosome amplification and multipolar divisions, leading to p53 stabilization and apoptosis of epidermal progenitors. The resulting delayed epidermal stratification led to skin barrier defects. Plk4 transgene expression was shut down postnatally in the surviving mice and PLK4OE mice never developed skin tumours. Concomitant PLK4OE and p53 deletion (PLK4OE/p53cKO) rescued the differentiation defects, but did not prevent the apoptosis of PLK4OE cells. Remarkably, the short-term presence of cells with supernumerary centrosomes in PLK4OE/p53cKO mice was sufficient to generate aneuploidy in the adult epidermis and triggered spontaneous skin cancers with complete penetrance. These results reveal that aneuploidy induced by transient centrosome amplification can accelerate tumorigenesis in p53-deficient cells.

New insights into centrosome imaging in Drosophila and mouse neuroepithelial tissues.

The centrosome is the main microtubule-organizing center in animal cells. It participates in the assembly of a bipolar spindle that ensures accurate segregation of chromosomes during mitosis. Recently, mutations in centrosome genes have been identified in patients affected by neurodevelopmental disorders. In fact, the etiology of several neurodevelopmental pathologies seems to be linked to defects in the assembly of the mitotic spindle in the neural stem cell compartment during neurogenesis. Therefore, getting better insights into the structure and function/dysfunction of the mitotic spindle apparatus in an intact tissue environment is of utmost importance. However, imaging nanometer-scale structures like centrosomes and microtubule bundles within the depth of a tissue is still challenging. Here we describe two procedures to acquire high-resolution images on fixed tissues and to perform live imaging of microtubule-based structures in the neuroepithelia of the Drosophila brain and of the mouse neocortex. We take advantage of the accumulation of centrosomes and mitotic figures at the apical surface of these polarized tissues to improve the quality of staining and imaging. Both Drosophila and mouse models with centrosome dysfunction showed abnormalities in the neuroepithelium reminiscent of the ones described in brains of human patients. These observations have highlighted their value as model organisms to study the etiology of human neurodevelopmental pathologies.

Microcephaly: STIL(l) a tale of too many centrosomes.

Centrosome mutations associated with microcephaly are normally thought to result in loss-of-function phenotypes. A new study shows, however, that mutations found in the human microcephaly STIL gene cause centrosome amplification, suggesting a direct link between the presence of extra centrosomes and the establishment of this disease.

Centrosome amplification causes microcephaly.

Centrosome amplification is a hallmark of human tumours. In flies, extra centrosomes cause spindle position defects that result in the expansion of the neural stem cell (NSC) pool and consequently in tumour formation. Here we investigated the consequences of centrosome amplification during mouse brain development and homeostasis. We show that centrosome amplification causes microcephaly due to inefficient clustering mechanisms, where NSCs divide in a multipolar fashion producing aneuploid cells that enter apoptosis. Importantly, we show that apoptosis inhibition causes the accumulation of highly aneuploid cells that lose their proliferative capacity and differentiate, thus depleting the pool of progenitors. Even if these conditions are not sufficient to halt brain development, they cause premature death due to tissue degeneration. Our results support an alternative concept to explain the etiology of microcephaly and show that centrosome amplification and aneuploidy can result in tissue degeneration rather than overproliferation and cancer.

Never tear us apart--the importance of centrosome clustering.

The presence of more than two centrosomes (centrosome amplification) at the onset of mitosis has long been associated with multipolar spindle formation, and with the generation of genetic instability. However, in recent years, several studies have shown that a process termed 'centrosome clustering' actively contributes to bipolar division by promoting the gathering of extra centrosomes in two main poles. In this Commentary, we describe the main proteins that are involved in centriole duplication and discuss how centrosome amplification can be generated both in vitro and in vivo. We then summarize what is currently known about the processes that contribute to bipolar spindle formation when extra centrosomes are present, and which forces contribute to this process. Finally, we discuss how extra centrosomes might contribute to tumorigenesis, giving emphasis to the role of centrosome amplification in promoting genetic instability.

Adhesion molecules in the stem cell niche--more than just staying in shape?

The expression of adhesion molecules by stem cells within their niches is well described, but what is their function? A conventional view is that these adhesion molecules simply retain stem cells in the niche and thereby maintain its architecture and shape. Here, we review recent literature showing that this is but one of their roles, and that they have essential functions in all aspects of the stem cell-niche interaction--retention, division and exit. We also highlight from this literature evidence supporting a simple model whereby the regulation of centrosome positioning and spindle angle is regulated by both cadherins and integrins, and the differential activity of these two adhesion molecules enables the fundamental stem cell property of switching between asymmetrical and symmetrical divisions.

beta1 integrin maintains integrity of the embryonic neocortical stem cell niche.

During embryogenesis, the neural stem cells (NSC) of the developing cerebral cortex are located in the ventricular zone (VZ) lining the cerebral ventricles. They exhibit apical and basal processes that contact the ventricular surface and the pial basement membrane, respectively. This unique architecture is important for VZ physical integrity and fate determination of NSC daughter cells. In addition, the shorter apical process is critical for interkinetic nuclear migration (INM), which enables VZ cell mitoses at the ventricular surface. Despite their importance, the mechanisms required for NSC adhesion to the ventricle are poorly understood. We have shown previously that one class of candidate adhesion molecules, laminins, are present in the ventricular region and that their integrin receptors are expressed by NSC. However, prior studies only demonstrate a role for their interaction in the attachment of the basal process to the overlying pial basement membrane. Here we use antibody-blocking and genetic experiments to reveal an additional and novel requirement for laminin/integrin interactions in apical process adhesion and NSC regulation. Transient abrogation of integrin binding and signalling using blocking antibodies to specifically target the ventricular region in utero results in abnormal INM and alterations in the orientation of NSC divisions. We found that these defects were also observed in laminin alpha2 deficient mice. More detailed analyses using a multidisciplinary approach to analyse stem cell behaviour by expression of fluorescent transgenes and multiphoton time-lapse imaging revealed that the transient embryonic disruption of laminin/integrin signalling at the VZ surface resulted in apical process detachment from the ventricular surface, dystrophic radial glia fibers, and substantial layering defects in the postnatal neocortex. Collectively, these data reveal novel roles for the laminin/integrin interaction in anchoring embryonic NSCs to the ventricular surface and maintaining the physical integrity of the neocortical niche, with even transient perturbations resulting in long-lasting cortical defects.

Adherens junction domains are split by asymmetric division of embryonic neural stem cells.

Investigating the mechanisms controlling the asymmetric division of neocortical progenitors that generate neurones in the mammalian brain is crucial for understanding the abnormalities of cortical development. Partitioning of fate determinants is a key instructive step and components of the apical junctional complex (adherens junctions), including the polarity proteins PAR3 and aPKC as well as adhesion molecules such as N-cadherin, have been proposed to be candidate determinants. In this study, however, we found no correlation between the partitioning of N-cadherin and fate determination. Rather, we show that adherens junctions comprise three membrane domains, and that during asymmetrical division these are split such that both daughters retain the adhesive proteins that control cell position, but only one daughter inherits the polarity proteins along with the apical membrane. This provides a molecular explanation as to how both daughters remain anchored to the ventricular surface after mitosis, while adopting different fates.

A novel function for cadherin-11 in the regulation of motor axon elongation and fasciculation.

We previously observed that cadherin-11, a type II cadherin, is expressed in growing motor and sensory axons in the mouse embryo. Here, we assessed its functional involvement in the regulation of axon elongation and fasciculation by evaluating the activity of a specific cadherin-11 homophilic ligand, cad11-Fc (cadherin-11 extracellular region fused to Fc fragment of IgG), on the length and organization of motor axons outgrowing from embryonic ventral spinal cord explants. Cad11-Fc substrate enhanced axon growth and prevented interactions occurring between growing axons, providing evidences for a role of cadherin-11 in the control of growth cone progression. Comparison of cadherin-11 with N-cadherin, a type I cadherin concomitantly expressed by motor axons, revealed similarities in their functional properties, including the ability to reorganize the actin cytoskeleton through interactions with catenins, but differences in their axon growth-promoting activity, arguing for subtle differences in their contributions to peripheral nerve elongation.

N-cadherin activation substitutes for the cell contact control in cell cycle arrest and myogenic differentiation: involvement of p120 and beta-catenin.

N-cadherin is expressed throughout skeletal myogenesis and has been proposed to be involved in the differentiation program of myogenic precursors. Here, we further characterize the N-cadherin involvement and its mechanism of action at the onset of differentiation, through controlled N-cadherin activation by plating isolated C2 myoblasts on surfaces coated with a chimeric Ncad-Fc homophilic ligand (N-cadherin ectodomain fused to the immunoglobulin G Fc fragment). We show that N-cadherin activation substitutes for the cell density in myogenic differentiation by promoting myogenin and troponin T expression. In addition, N-cadherin adhesion participates to the associated cell cycle arrest through the nuclear accumulation of cyclin-dependent kinase inhibitors p21 and p27. Mouse primary myoblast cultures exhibited similar responses to N-cadherin as C2 cells. RNA interference knockdowns of the N-cadherin-associated cytoplasmic proteins p120 and beta-catenin produced opposite effects on the differentiation pathway. p120 silencing resulted in a decreased myogenic differentiation, associated with a reduction in cadherin-catenin content, which may explain its action on myogenic differentiation. beta-Catenin silencing led to a stimulatory effect on myogenin expression, without any effect on cell cycle. Our results demonstrate that N-cadherin adhesion may account for cell-cell contact-dependent cell cycle arrest and differentiation of myogenic cells, involving regulation through p120 and beta-catenins.

Lamellipodium extension and cadherin adhesion: two cell responses to cadherin activation relying on distinct signalling pathways.

Cell adhesion molecules of the cadherin family contribute to the regulation of cell shape and fate by mediating strong intercellular adhesion through Ca2+-dependent interaction of their ectodomain and association of their cytoplasmic tail to actin. However, the mechanisms co-ordinating cadherinmediated adhesion with the reorganization of the actin cytoskeleton remain elusive. Here, the formation of de novo contacts was dissected by spreading cells on a highly active N-cadherin homophilic ligand. Cells responded to N-cadherin activation by extending lamellipodium and organizing cadherin-catenin complexes and actin filaments in cadherin adhesions. Lamellipodium protrusion, associated with actin polymerization at the leading edge sustained the extension of cadherin contacts through a phosphoinositide 3-kinase (PI 3-kinase)-Rac1 pathway. Cadherin adhesions were formed by PI 3-kinase-independent, Rac1-dependent co-recruitment of adhesion complexes and actin filaments. The expression and localization of p120 at the plasma membrane, associated with an increase in membrane-associated Rac1 was required for both cell responses, consistent with a major role of p120 in signalling pathways initiated by cadherin activation and contributing to Rac1-dependent contact extension and maturation. These results provide additional information on the mechanisms by which cadherin coordinates adhesion with dynamic changes in the cytoskeleton to control cell shape and intercellular junction organization.

[Regulation of cell adhesion by the cadherin cytoplasmic domain]. / La régulation de l'adherence cellulaire par la région cytoplasmique des cadhérines.

Juxtacrine cell interactions associated to cadherin-mediated cell-cell adhesion play a major role in the organization and homeostasis of tissues. Here, we review the intracellular molecules and regulations controlling the formation of cell-cell contacts initiated by homophilic interactions of cadherin ectodomain. These regulations involve proteins associated to cadherin cytoplasmic tail, named catenins, their association to the actin cytoskeleton and the stability of these complexes at the cell membrane. The underlying molecular mechanisms, which participate in the formation of dynamic cell-cell contacts, are intensively investigated.

Cross talk between tetanus neurotoxin-insensitive vesicle-associated membrane protein-mediated transport and L1-mediated adhesion.

The membrane-trafficking pathway mediated by tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) in neurons is still unknown. We show herein that TI-VAMP expression is necessary for neurite outgrowth in PC12 cells and hippocampal neurons in culture. TI-VAMP interacts with plasma membrane and endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptors, suggesting that TI-VAMP mediates a recycling pathway. L1, a cell-cell adhesion molecule involved in axonal outgrowth, colocalized with TI-VAMP in the developing brain, neurons in culture, and PC12 cells. Plasma membrane L1 was internalized into the TI-VAMP-containing compartment. Silencing of TI-VAMP resulted in reduced expression of L1 at the plasma membrane. Finally, using the extracellular domain of L1 and N-cadherin immobilized on beads, we found that the silencing of TI-VAMP led to impaired L1- but not N-cadherin-mediated adhesion. Furthermore, TI-VAMP- but not synaptobrevin 2-containing vesicles accumulated at the site of the L1 bead-cell junction. We conclude that TI-VAMP mediates the intracellular transport of L1 and that L1-mediated adhesion controls this membrane trafficking, thereby suggesting an important cross talk between membrane trafficking and cell-cell adhesion.

Heterogeneity and regulation of cellular prion protein glycoforms in neuronal cell lines.

The normal cellular prion protein is a small sialoglycoprotein highly expressed in neurons, the physiological function of which is largely unknown. Due to extensive N-glycosylations with a wide range of oligosaccharides, the prion protein displays a complex glycosylation pattern that could be of relevance for its function. The cellular prion protein patterns in adult mouse and rat brain, and in neuronal cell lines, appeared highly heterogeneous, as distinct levels and glycoforms of cellular prion protein were revealed by immunoblotting of corresponding samples. Amongst neuronal cell lines, mouse N2a neuroblastoma cells expressed low levels of endogenous prion protein. Mouse hypothalamic GT1-7 cells and rat pheochromocytoma PC-12 cells expressed highly glycosylated forms of cellular prion protein that were found neither in adult mouse and rat brain, nor in mouse brain during development. In contrast, rat B104 neuroblastoma cells abundantly expressed N-glycosylated cellular prion protein forms similar to those observed in mouse and rat brain. In all these cell lines, the prion protein was normally exported to and expressed at the outer cell membrane. Our results suggest that B104 cells may represent an appropriate cell model to investigate the physiological role of cellular prion protein in further detail as they highly express the normal 'brain-like' cellular prion protein glycoforms. In addition, we observed that the various prion glycoforms in B104 cells were tightly regulated both as a function of cell density and during neuronal differentiation, implying a potential role of cellular prion protein in cell-cell interactions and differentiation.